B cells isolated from the B cell receptor (BCR) trangenic model AM14 recognize a prototypic autoantigen, IgG2a with relatively low affinity, and are relatively unresponsive to most IgG2a-containing immune complexes (IC). However, these rheumatoid factor (RF) producing B cells proliferate vigorously in response to IC consisting of IgG2a bound to chromatin (chromatin-IC) via a mechanism that involves sequential engagement of the BCR located on the plasma membrane and Toll-like receptor 9 (TLR9) located in an internal compartment. Chromatin (and other protein/nucleic acid molecular entities) are prominent autoantibody targets in SLE and preliminary data suggests that this sequential activation paradigm, established for the activation of RF+ B cells, may also apply to the activation of other autoantigen reactive B cells. Further evaluation of the functional outcome of this activation pathway is necessary in order to determine whether there are unique features that distinguish the activation of autoreactive B cells from cells signaled through either the BCR or TLR9 alone. It will also be critical to determine whether the same activation mechanism applies to non-RF autoreactive B cells, and if so, whether this route of activation provides a unique therapeutic target. These issues will be addressed through the following specific aims: 1. Define the biochemical properties of immunostimulatory chromatin/DNA by using IC containing purified chromatin fractions and defined DNA fragments; monitor the binding, uptake and persistence of these DNA fragment IC; 2. Directly compare functional properties of B cells activated by BCR engagement alone, TLR engagement alone, or BCR/TLR9 sequential engagement with regard to parameters such as lipid raft formation, expression of specific cell surface differentiation antigens, antibody and cytokine production, sensitivity to pro-apoptotic effects of Fas-ligand, sensitivity to factors produced by autoreactive T cells and or dendritic cells, and capacity to home to particular lymphoid microenvironments and then survive in vivo; 3. Determine to what extent the BCR/TLR sequential engagement paradigm applies to autoantibody responses in general by stimulating AM14 RF+ B cells in vitro with non-chromatin-associated autoantigens and by monitoring autoantibody production in TLR-deficient autoimmune-prone mice; and 4. Determine whether the development and/or progression of SLE can be blocked by inhibitors of the TLR9 signaling pathway. The results of the studies will provide important information regarding the development of novel therapies for the treatment of systemic diseases such as SLE.